Pycnopulvinus aurantiacus gen. et sp. nov., a new sporocarp-forming member of Pucciniomycotina

MycoKeys 8: 43-50 (20 | 4) A peer-reviewed open-access journal
doi: 10.3897/mycokeys.8.7676 RESEARCH ARTICLE O Myco Keys

http://mycokeys.pensoft.net Launched to accelerate biodiversity research

Pycnopulvinus aurantiacus gen. et sp. nov., a new
sporocarp-forming member of Pucciniomycotina

Merje Toome', M. Catherine Aime'

| Department of Botany and Plant Pathology, Purdue University, 915 W. State St., West Lafayette, IN 47907, USA

Corresponding author: M. Catherine Aime (maime@purdue.edu)

Academic editor: D. Begerow | Received 15 April 2014 | Accepted 29 October 2014 | Published 3 November 2014

Citation: Toome M, Aime MC (2014) Pycnopulvinus aurantiacus gen. et sp. nov., a new sporocarp-forming member of
Pucciniomycotina. MycoKeys 8: 43-50. doi: 10.3897/mycokeys.8.7676

Abstract

An unusual fungus producing minute orange stilboid sporocarps was found on a palm leaf mid-rib in a
Neotropical forest. Morphological observations could not place this collection into any previously de-
scribed species or genus and, due to an absence of sexual structures, even higher level placement was
uncertain. Phylogenetic analysis of a portion of the large subunit and the internal transcribed spacer of
the nuclear ribosomal DNA indicated that this fungus is related to Heterogastridium pycnidioideum and
belongs to Heterogastridiales, Microbotryomycetes (Pucciniomycotina). A new genus and species, Pycnop-

ulvinus aurantiacus, are proposed here to accommodate this fungus.

Key words
Ceratocystis, Fungal biodiversity, litter fungi, palm fungi, Pycnobasidium, tropical mycology

Introduction

The majority of Pucciniomycotina (Basidiomycota) species have life cycles that include
the production of microscopic fruiting structures (e.g., spermogonia of rust fungi), but
only a few species within the subphylum form macroscopic fruiting bodies (Swann et
al. 2001; Aime et al. 2014). Although these sporocarps vary in form (e.g., see Aime et
al. 2014), many are stilboid or pycnidioid with spore masses produced at the base of
the sporocarp and exiting through the tip of the neck. Fungi with such fruiting bodies
can be found mostly in Atractiellomycetes (Oberwinkler and Bandoni 1982) and, as is
true for many other members of Pucciniomycotina, little is known about their biology
or biodiversity.

Copyright Merje Toome, M. Catherine Aime. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

44 Merje Toome & M. Catherine Aime / MycoKeys 8: 43-50 (2014)

Heterogastridium pycnidioideum Oberw. & R. Bauer (anamorph Hyalopycnis
blepharistoma (Berk.) Seeler) is one such fungus that produces stilboid fruiting bod-
ies. It is a strictly filamentous species with simple septal pores and specialized orga-
nelles—colacosomes—associated with mycoparasitism (Oberwinkler et al. 1990). Het-
erogastridium is placed in Heterogastridiaceae and Heterogastridiales (Oberwinkler et
al. 1990), which are now placed in Microbotryomycetes (Weiss et al. 2004). Bauer et
al. (2006) placed three genera in Heterogastridiaceae in addition to Heterogastridum—
Atractocolax R. Kirschner, R. Bauer & Oberw., Colacogloea Oberw. & R. Bauer, and
Krieglsteinera Pouzar. Of these, neither sequence data nor reference cultures are availa-
ble for two of the genera, Atractocolax and Krieglsteinera, both of which are monotypic.
Colacogloea, on the other hand, has been demonstrated several times to form a separate
clade of Microbotryomycetes, distant from Heterogastridiales (e.g. Aime et al. 2006;
Kurtzman et al. 2011). Thus, at present, only Heterogastridium and its sole species, H.
pycnidioideum, can be confidently assigned to Heterogastridiales with molecular data.

An unusual stilboid fungus was discovered on the mid-rib of a palm leaf in lit-
ter that could not be confidently assigned to any previously described genus. DNA
sequence data and phylogenetic analyses indicated that the collection represents a new
member of Heterogastridiales. Herein we describe and illustrate Pycnopulvinus auran-
tiacus gen. et sp. nov. and provide a phylogenetic analysis of Heterogastridiales.

Methods

The specimen (PUL F2679) was collected near Bilsa Biological Station in Ecuador, in
the vicinity of N0.350444, W79.732075, on 3 May 2004, where it was growing on the
mid-rib of a dead palm leaf in the litter. The fungus was photographed and described
in the field after which small pieces of substrate bearing the fruiting bodies were dried
on an herbarium drier. Color designations refer to Kornerup and Wanscher (1978).
Duplicates are deposited in the Kriebel Herbarium at Purdue University (PUL) and the
herbarium of the Pontificia Universidad Catolica del Ecuador (QCA).

Morphological characters of the fruiting bodies were observed first with an Olym-
pus SZ61 dissecting microscope. With the aid of the dissecting microscope, a few
fruiting bodies were carefully removed from the substrate and placed in a sterile water
droplet on a microscope slide, permitting five minutes of rehydration before further
preparation. Thereafter microscopic characters were examined with a Nikon Eclipse
80i microscope with standard differential interference contrast (DIC) settings and
with 10x, 20x, 40x and 100x objectives. The length and width of 20 spores was meas-
ured from three different fruiting bodies with an ocular micrometer using 100x oil-
immersion objective. Images were taken with Nikon Digital Sight DS-Fil camera
setup and measurements were calibrated with a stage micrometer.

For molecular characterization, five dry fruiting bodies were carefully removed
from the leaf surface to avoid the inclusion of leaf material and potential contami-

nants. These were used for DNA extraction with the E.Z.N.A. High Performance

Pycnopulvinus aurantiacus gen. et sp. nov., a new sporocarp-forming member... 45

DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA), following the manufacturers’
instructions for samples with lower DNA content (protocol 3). PCR reactions were
carried out in 25 uL reactions that contained 12.5 wL of Apex Taq RED Master Mix
(Genesee Scientific, San Diego, CA, USA), 1.25 uL of each primer (10 pM), 5 pL of
molecular grade water and 5 uL of template DNA. Amplification of the ITS region
was conducted with primer pair ITS1F (Gardes and Bruns 1993) and ITS4 (White
et al. 1990), and the LSU region was amplified with LROR and LR5 (Vilgalys and
Hester 1990). Amplification conditions followed Toome et al. (2013). Sequencing of
amplified fragments was performed by Beckman Coulter, Inc. (Danvers, MA), using
the same primers that were used for amplification. Sequences were edited with Se-
quencher 5.2.3 (Gene Codes Corporation, Ann Arbor, MI, USA) and are deposited
in GenBank.

A BLASTn analysis in GenBank (http://www.ncbi.nlm.nih.gov) was used to lo-
cate similar sequences for phylogenetic analyses. For the LSU dataset, sequences shar-
ing >92% identity with PUL F2679 were selected and for the ITS dataset sequences
sharing >85% identity with PUL F2679 were included. Sequences from Rhodotorula
hylophila and R. javanica were added based on their relatedness according to the analy-
ses of Kurtzman et al. (2011) and R. yarrowii was included for rooting purposes. Only
the sequences from type strains were retained for previously described species. Acces-
sion numbers for sequences used are indicated on Figure 1.

Both datasets were aligned separately using the Muscle algorithm in MEGA 5.2
(Kumar et al. 2008), yielding 695 bp and 605 bp alignments for the ITS and the par-
tial LSU region, respectively. These two alignments were subsequently concatenated
for phylogenetic analyses. The final alignment is available in TreeBASE (http://tree-
base.org) under accession number 15679.

Phylogenetic analyses were performed via the CIPRES Science Gateway (Miller et
al. 2010). The maximum likelihood (ML) analyses were conducted in RAxML-HPC2
7.6.3 using the -k option for bootstrap analysis. Bayesian posterior probability analy-
ses were conducted with MrBayes 3.2.2 with parameters set to 10 000 000 genera-
tions, two runs and four chains. The resulting phylogenetic tree was edited in Incscape

(http://www.inkscape.org).

Results

Sporocarps of PUL F2679 had a swollen cushion-like basal region, measuring 0.2 to 1
mm in diameter after drying. The basal region was pigmented, ranging from light to
dark orange when fresh and appearing orange-brown after drying. This base supports a
narrow synnemata-like structure with a 1 to 2 mm long neck. At the apex of the neck, a
light yellow to orange mucous droplet of spores is formed (Figure 2 a-f). While the size
of sporocarps was variable, all of them produced a spore-containing droplet. Although
it could not be definitely determined, the spores appear to be asexually produced.
Also, despite numerous attempts to isolate the fungus into pure culture from revived

46 Merje Toome & M. Catherine Aime / MycoKeys 8: 43-50 (2014)

Rhodotorula hylophila CBS 6226" (AF363645; AF444622)
Rhodotorula javanica CBS 5236" (AF189935; AF444532)

Rhodotorula yarrowii CBS 7417' (AF189971; AF444628)

Figure |. Maximum likelihood tree illustrating the placement of Pycnopulvinus aurantiacus in relation
to other known members of Heterogastridiaceae. The analysis was performed with combined LSU and
ITS sequence data and the topology was rooted with Rhodotorula yarrowii. The numbers above and below
branches show the bootstrap and posterior probability values, respectively. LSU and ITS GenBank acces-
sion numbers of each used strain are given in the brackets. Superscript letter T indicates sequence data
that originate from the type.

spores, no isolate was obtained. Only an isolate of Ceratocystis paradoxa (deposited in
GenBank as AY821864; CBS 116770) was frequently recovered during these attempts.

Of previously sequenced species, PUL F2679 shared the most sequence identity
with H. pycnidioideum (= H. blepharistoma) at 92% (414 bp of 452 bp) in the LSU
region and 86% (515 bp of 596 bp) in the ITS region. The only other close match
was to a previously sequenced but undescribed isolate, CBS 196.95 (GenBank no.
LSU—AY323905; ITS—AY323904), which shared 98% identity (446 bp of 453 bp)
in the LSU region and 99% identity (609 bp of 616 bp) in the ITS region. Results of

phylogenetic analyses are presented in Figure 1.

Taxonomy

Pycnopulvinus Toome & Aime, gen. nov.
MycoBank MB808523

Diagnosis. Member of Heterogastridiaceae, Heterogastridiales, Microbotryomycetes,
Pucciniomycotina. Pycnopulvinus is similar to Heterogastridium, but differs in possessing
a distinct basal cushion, segmented spores, and pigmented sporocarps. Pycnopulvinus
can also be distinguished with rDNA sequence data.

Type. Pycnopulvinus aurantiacus Toome & Aime

Description. Minute, pigmented stilboid sporocarps with a swollen basal region
and long tubular neck bearing a mucoid droplet of spores at the tip.

Ecology and distribution. On palm litter in South America (Ecuador); known
from sequence data in Central America (Costa Rica).

Pycnopulvinus aurantiacus gen. et sp. nov., a new sporocarp-forming member... 47

Etymology. pycno- = dense, compact, and pulvinus = cushion, for the distinctive
cushion-like base of the sporocarp.

Discussion. ‘The genus is closely related to Heterogastridium, but has orange-color-
ed fruiting bodies that are larger and form a distinct basal cushion. Of the other three
genera currently recognized in Heterogastridiales (Kirk et al. 2008, Bauer et al. 2006),
Pycnopulvinus can be readily separated from Colacogloea on the basis of DNA sequence
data (Aime et al. 2006), and the absence of stilboid fructifications or septate spores in
the latter, from Krieglsteinera which does not form fruiting bodies or multicelled spores
and parasitizes ascocarps, and from Atractocolax which forms smaller (140 um diam.),
gelatinous, hyaline sporocarps, produces unicellular spores and is associated with bark
beetles. Currently, Pycnopulvinus is a monotypic genus. A previously undescribed iso-
late, CBS 176.95 (isolated from the tropical forest of Costa Rica and accessioned in
the GenBank as “Pycnobasidium sp.” — a generic name that has not been validly pub-
lished — appears to represent a member of this genus. In the absence of morphological
or other additional data, it cannot be determined whether CBS 176.95 is conspecific
with P. aurantiacus or represents a second species of Pycnopulvinus. Despite the rapid
accumulation of environmental sequencing data, no studies thus far have published
sequences referable to Pycnopulvinus, indicating that members of the genus are prob-
ably not widely dispersed in commonly sampled habitats.

Pycnopulvinus aurantiacus Toome & Aime, sp. nov.
MycoBank MB808524
Figure 2

Diagnosis. Sporocarps minute, orange, with a swollen basal cushion (up to 3 mm
wide) and long narrow neck (up to 3 mm long) subtended by a light yellow to orange
mucous droplet (up to 1.5 mm diam.) of hyaline, 2—4 celled spores, averaging 3.25 x
11.8 um. Found on palm leaf litter.

Type. ECUADOR. Manabi Division, near Bilsa Biological Station, in the vicinity
of N0.350444, W79.732075, on palm leaf mid-rib in the litter, 3 May 2004, M.C.
Aime, MCA 2548 (holotype PUL F2679; isotype QCA). GenBank no. KJ676978
(ITS), KJ676979 (LSU).

Description. Gregarious, light to dark orange (ca. 5A6—7), superficial, stilboid spo-
rocarps with swollen globose base (0.5—3 mm wide in fresh specimen, drying to 0.2-1
mm), surrounded by hyphae with globular apical cells, 20-30 um wide and 45-55 um
long. Sporocarp necks erect, long (0.5—2 mm), narrow (up to 110 um at base, 50-70
um at middle and widening up to 130 pum at tip), tubular, light yellow to orange,
smooth. Hyphae on the outer layer of the neck 5 um wide, septate; hyphae at the base
and inside the neck 2—2.5 um wide, septate. Ostiolar hyphae extend from the outer lay-
er of the neck cells, hyaline, non-septate, 10-12 um wide, narrowing at the tip. Clamp
connections not observed. Spores accumulate in pale to orange mucous droplets at tips

48 Merje Toome & M. Catherine Aime / MycoKeys 8: 43-50 (2014)

4
wa! \

ny
f

,
y

ba TSR a “
Figure 2. Pycnopulvinus aurantiacus (holotype PUL F2679). a Field photo of fresh sporocarps on palm
leaf. Note the variable size and color of the sporocarps. Bar = 2 mm b-d Sporocarps of various stages
and sizes after drying. Bars = 0.5 mm e Sporocarp as seen under the light microscope. Note the swol-
len pigmented base with surrounding large globose cells and the long tubular neck with a widening tip.
Bar = 200 um f Tip of the neck with spore mass exiting the sporocarp. Bar = 25 um g Ostiolar hyphae at
the tip of the neck. Bar = 10 um h Outer surface of the neck, wide hyphae are visible, septa are marked
with arrows. Bar = 10 um i Globose cells (marked with asterisk) surrounding the base of the sporocarp.
Bar = 25 um j Multicellular spores produced inside the sporocarps. Note the four-celled spores on the left

a

breaking into smaller compartments. Bar = 10 pm.

of sporocarp necks, approximately half of the size of the basal cushion in diam. (up to
1.5 mm diam. in fresh material). Spores hyaline, mostly 2—4 celled and cylindrical or
somewhat fusiform, measuring 3—4 x 7-18 um (average 3.25 x 11.8 um), breaking into
smaller compartments. No sporogenous cells were detected at the interior of the sporo-
carp, but the spores are likely asexually produced. Basidia not observed.

Pycnopulvinus aurantiacus gen. et sp. nov., a new sporocarp-forming member... 49

Ecology and distribution. On palm leaf litter in tropical forest of Ecuador. Pos-
sibly occurring in association with other fungi. Known only from the type locale.

Etymology. aurantiacus = orange, for the color of fresh sporocarps.

Specimens examined. PUL F2679.

Discussion. The sporocarps most likely represent an asexual stage of P. auran-
tiacus. Anamorphic stilboid conidiomata have been described for other members of
Pucciniomycotina, especially in Atractiellomycetes (e.g. Oberwinkler et al. 2006).
However, multicellular spores have not previously been described for any sporocarp-
forming member of Atractiellomycetes or Microbotryomycetes. ‘The organic matter of
palm trees is not a common substrate among Pucciniomycotina and only one other
basidiocarp-forming genus, Agaricostilbum Wright, is known to specifically inhabit
palm litter (Wright et al. 1981). The recovery of C. paradoxa from the material might
hint on a possible mycoparasitic strategy for P. aurantiacus.

Acknowledgements

We are grateful to Prof. Rosario Briones of Pontificia Universidad Catolica del Ecua-
dor, Quito for facilitating specimen deposition and exchanges at Pontificia Universi-
dad Catolica del Ecuador (QCA), to Drs. Harry Evans and Gary Samuels for facilitat-
ing permitting and collecting logistics in Ecuador and companionship in the field, and
the staff and volunteers at Bilsa. Cindy Park is gratefully acknowledged for laboratory
assistance at the USDA. We are thankful to Tom Creswell, Gail Ruhl and Anna Meier
from the Purdue University Plant and Pest Diagnostic Laboratory for use of micros-
copy facilities and to Mehrdad Abbasi assisting with submission to Kriebel Herbarium
at Purdue. We are also thankful to the two anonymous reviewers for constructive com-
ments and suggestions on an earlier version of this manuscript.

References

Aime MC, Matheny PB, Henk DA, Frieders EM, Nilsson RH, Piepenbring M, McLaughlin
DJ, Szabo LJ, Begerow D, Sampaio JP, Bauer R, Weiss M, Oberwinkler F, Hibbett DS
(2006) An overview of the higher level classification of Pucciniomycotina based on combined
analyses of nuclear large and small subunit rDNA sequences. Mycologia 98: 895-905. doi:
10.3852/mycologia.98.6.896

Aime MC, Toome M, McLaughlin DJ (2014) Pucciniomycotina. In: McLaughlin DJ, Spatafora
JW (Eds) The Mycota: Systematics and Evolution, 2” edition, vol 7A. Springer-Verlag, Berlin,
271-294.

Bandoni RJ, Oberwinkler F (1981) Ayalopycnis blepharistoma: a pycnidial basidiomycete. Ca-
nadian Journal of Botany 59: 1631-1620. doi: 10.1139/b81-219

Bauer R, Begerow D, Sampaio JP, Weiss M, Oberwinkler F (2006) The simple-septate basidi-
omycetes: a synopsis. Mycological Progress 5: 41-66. doi: 10.1007/s11557-006-0502-0

50 Merje Toome & M. Catherine Aime / MycoKeys 8: 43-50 (2014)

Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes: appli-
cation to the identification of mycorrhiza and rusts. Molecular Ecology 2: 113-118. doi:
10.1111/j.1365-294X.1993.tb00005.x

Kornerup A, Wanscher JH (1978) Methuen handbook of colour, 3" ed. Richard Clay Ltd.,
Chichester.

Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: A biologist-centric software for evolu-
tionary analysis of DNA and protein sequences. Briefings in Bioinformatics 9: 299-306.
doi: 10.1093/bib/bbn017

Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts, a Taxonomic Study, 5" edition, vol 3.
Elsevier, London, UK.

Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for infer-
ence of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments
Workshop. New Orleans, Louisiana, 1-8.

Oberwinkler F, Bandoni RJ (1982) A taxonomic survey of the gasteroid, auricularioid Heter-
obasidiomycetes. Canadian Journal of Botany 56: 1726-1750.

Oberwinkler F, Bauer R, Bandoni RJ (1990) Heterogastridiales: a new order of basidiomycetes.
Mycologia 82: 48-58. doi: 10.2307/3759962

Oberwinkler F, Kirschner R, Arenal F, Villarreal M, Rubio V, Begerow D, Bauer R (2006)
Two new pycnidial members of the Atractiellales: Basidiopycnis hyalina and Proceropycnis
pinicola. Mycologia 98: 637-649. doi:10.3852/mycologia.98.4.637

Swann EC, Frieders EM, McLaughlin DJ (2001) Urediniomycetes. In: McLaughlin DJ,
McLaughlin EJ, Lemke P (Eds) The Mycota: Systematics and Evolution, vol 7B. Springer-
Verlag, Berlin, 37-56.

Toome M, Roberson RW, Aime MC (2013) Meredithblackwellia eburnea gen. et sp. nov., Krie-
geriaceae fam. nov. and Kriegeriales ord. nov. — toward resolving higher-level classification
in Microbotryomycetes. Mycologia 105: 486-495. doi: 10.3852/12-251

Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically am-
plified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172:
4238-4246. http://jb.asm.org/content/172/8/4238.full.pdf+html

Weiss M, Bauer R, Begerow D (2004) Spotlights on heterobasidiomycetes. In: Agerer R, Piepen-
bring M, Blanz P (Eds) Frontier in Basidiomycote Mycology. IHW-Verlag, Eching, 7—48.

White TJ, Bruns TD, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White T]
(Eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego,
California, 315-322. doi: 10.1016/B978-0-12-372180-8.50042-1

Wright JE, Bandoni R, Oberwinkler F (1981) Agaricostilbum: an auricularioid basidiomycete.
Mycologia 73: 880-886. doi: 10.2307/3759799